Background Identification of genetic defects and elucidation of underlying pathways of photoreceptor degeneration constitute an essential step in developing targeted therapeutic strategies for retinal neurodegenerative diseases. N-NRL collaborates with numerous clinicians and scientists to identify genes associated with RP, LCA and syndromic diseases involving photoreceptor dysfunction. Results 1. Human retinal diseases We have developed an in-house pipeline for disease gene identification in retinopathies by exome sequencing (4). Using this approach in a two-generation family with early form of dominant macular dystrophy, we have identified a novel heterozygous segregating mutation in the ECM gene FBN2 that we are further characterizing. Our data point to a role for FBN2 in macular pathology and suggests an important link between rare and common forms of maculopathy. 2. Animal models of human retinal disease By GFP tagging of newborn rods and BrdU birthdating, we demonstrated that additional S-cones in the rd7 mouse retina originate from post-mitotic early-born photoreceptor precursors that are committed to rod fate and not by proliferation of cone photoreceptors as suggested earlier. We have further rescued the rd7 phenotype by transgenic expression of Nr2e3 in photoreceptor precursors under control of Crx or Nrl promoters, resulting in suppression of cone genes and restoration of rod morphology/function (1). Using the retinal degeneration slow (rds) mouse model and after global gene expression profiling in the retina and rod photoreceptors, we found that major defects in photoreceptor cell structure induce early homeostatic responses, which may function in a protective manner to promote cell viability. We identified a single key gene (Egr1) dysregulated in a sustained fashion in rds rod photoreceptors and retina and hypothesized that activation of Egr1 and neurotrophic factors may represent a protective immune mechanism contributing to slow retinal degeneration of the rds mouse model (7). We have generated mouse models of retinitis pigmentosa GTPase regulator (RPGR) to delineate its in vivo function and examine various treatment paradigms (3). We have characterized the rd9 mouse carrying ORF15 mutation (8), developed a conditional knockout, and are in the process of generating a knockin model of human RPGR-G173R mutation to evaluate disease pathology. Retinal degeneration is commonly observed in syndromic ciliopathies and CEP290 mutations are detected in most forms (including LCA). Thus, we have focused on functional analysis of CEP290 and two of its interacting proteins: centrosomal protein CC2D2A, mutations in which cause Joubert and Meckel-Gruber syndrome, and CP110 that antagonizes CEP290 in cilia formation. We have discovered interaction of CEP290 with MKKS BBS6, mutations in which cause Bardet-Biedl Syndrome (BBS). However, combining the Cep290rd16 allele with loss of Mkks results in an improved sensory cilia phenotype in retina, cochlea, and olfactory epithelium (5). Rescue of the disease phenotype resulting from mutations in one ciliary protein by eliminating an interacting protein introduces a novel therapeutic approach for treating ciliopathies. We have also produced Cep290-/- and Cep290gt/gt (gene trap with N-terminal truncation) mice that exhibit different and more severe phenotypes than the Cep290rd16/rd16 mice. Evaluation of the Cep290-/- mouse retina reveals an essential role of CEP290 in the elaboration of connecting cilium and outer segments. To complement cell biological findings, we have initiated a study of over 250 common small metabolites (with Metabolon) in P2 and P10 retina from Cep290rd16 (and Pde6b/rd1) mice. Biochemical analyses will be compared with gene profile analysis of P10 Cep290-/- retina to understand early changes that precede degeneration. Cc2d2a-/- mice exhibit embryonic lethality, with extensive developmental defects that include heterotaxy, liver fibrosis, anophthalmia, hydrocephalus and polydactyly. Occasional Cc2d2a-/- animals that survive up to one month display severe hydrocephalus and retinal dystrophy. Our studies suggest that Cc2d2a function is critical for normal body plan and organ development and may involve cilia-mediated signaling pathways. Furthermore, we have generated Cp110 floxed mice that will be bred to Zp3-Cre, Rx-Cre and Crx-Cre lines to create complete null and retina-specific knockout lines. The Nrl-/- mouse carries a retina with predominantly S-opsin containing cones that exhibit molecular and functional characteristics of WT cones. We observed that Nrl-/- retina undergoes a rapid but transient period of degeneration in early adulthood. By four months of age, cone degeneration stabilizes, resulting in a thinned but intact outer nuclear layer with residual cones expressing S- and M-opsins and a preserved photopic ERG. Combining whole-transcriptome analysis by Affymetrix microarray and by Next Generation Sequencing (RNA-Seq) of 1, 2, and 4 month old Nrl-/- retina, we identified 39 common differentially expressed genes, converging on tumor necrosis factor (Tnf) as a central hub, and underscoring the importance of immune response in cone death observed in Nrl-/-retina (6) We have identified a new mouse mutant (collaboration with B Chang, Jax Labs) displaying complete absence of rod photoreceptors, immature cone-like photoreceptors, and congenital blindness. The mouse carries a novel frame-shift deletion in Crx that leads to a longer translated protein, similar to those described in patients with LCA or cone-rod dystrophy (CRD). Since Crx-/- mice do not replicate the human phenotype, our goal is to validate this mouse as a new model for LCA and CRD associated with Crx mutation and to use it for gene therapy. Furthermore, we established that the extended CRX protein does not bind NRL and acts as a dominant negative. Thus, this mutant could be very valuable also to study Crx function during retinal development. 3. Altered homeostasis in the aging retina Despite the link with oxidative stress, mitochondria function and protein metabolism, precise molecular pathways leading to rod loss in aging are not delineated. By microarray and RNA-Seq profiling of aging mouse rods, we validated changes in mitochondrial gene expression. We further examined mitochondrial function in retina from ad libitum and caloric-restricted mice at 6, 12, 16 and 21 months of age (collaboration with Wei Li and Robert Balaban). At the same time, we are collecting human retina, RPE and choroid at different ages and from normal and disease individuals to correlate genotype with aging-associated expression changes (collaboration with Goncalo Abecasis). 4. In vitro models of human retinal disease Reprogramming of somatic cells to a pluripotent state allows de novo differentiation to photorecetors and makes it possible to model retinal disease in a dish using the patients own cells to study of disease pathogenesis and discovery of small molecules for therapy. We are initially focusing on patients with mutations in CEP290 that present LCA or Joubert syndrome phenotypes. Provisions for recruitment of these patients are included in human clinical protocols led by Brian Brooks and Meral Gunay-Aygun. Sam Jacobson and Robert Koenekoop are recruiting additional patients. Significance Our human genetic studies have identified a number of new disease genes. Our work in animal models has highlighted an interesting convergence on biogenesis and transport functions associated with primary cilia and revealed novel pathways contributing to photoreceptor homeostasis. These results will have significance for treating patients with LCA and retinal degeneration.